Secondary literature sources for the Brr6_like_C_C domain

For each of the hand selected primary papers associated with the Brr6_like_C_C domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Vesicle trafficking maintains nuclear shape in Saccharomyces cerevisiae during membrane proliferation.

Webster MT, McCaffery JM, Cohen-Fix O
J Cell Biol. 2010; 191: 1079-88

The parameters that control nuclear size and shape are poorly understood. In yeast, unregulated membrane proliferation, caused by deletion of the phospholipid biosynthesis inhibitor SPO7, leads to a single nuclear envelope "flare" that protrudes into the cytoplasm. This flare is always associated with the asymmetrically localized nucleolus, which suggests that the site of membrane expansion is spatially confined by an unknown mechanism. Here we show that in spo7Delta cells, mutations in vesicle-trafficking genes lead to multiple flares around the entire nucleus. These mutations also alter the distribution of small nucleolar RNA-associated nucleolar proteins independently of their effect on nuclear shape. Both single- and multi-flared nuclei have increased nuclear envelope surface area, yet they maintain the same nuclear/cell volume ratio as wild-type cells. These data suggest that, upon membrane expansion, the spatial confinement of the single nuclear flare is dependent on vesicle trafficking. Moreover, flares may facilitate maintenance of a constant nuclear/cell volume ratio in the face of altered membrane proliferation.

Fad24, a mammalian homolog of Noc3p, is a positive regulator in adipocyte differentiation.

Tominaga K, Johmura Y, Nishizuka M, Imagawa M
J Cell Sci. 2004; 117: 6217-26

Adipocyte differentiation is controlled by complex actions involving gene expression and signal transduction. From metaphase to anaphase, peroxisome proliferator-activated receptor gamma, the CCAAT/enhancer-binding protein family and sterol regulatory element-binding protein-1 are known to function as master regulators. However, the mechanism underlying the earliest step, which triggers the initiation of differentiation, remains unknown. In previous reports, we have isolated a number of genes, whose expression increases in the early stage of differentiation in the mouse 3T3-L1 preadipocyte cell line. Here we report the cloning of the full-length cDNA and characterization of an unknown gene isolated previously and named fad24 (factor for adipocyte differentiation 24). Fad24 encodes a protein consisting of 807 amino acids. The deduced amino acid sequence was shown to have a basic leucine zipper motif and a NOC domain. Expression of fad24 was rapidly induced after stimulation with inducers. Furthermore, overexpression of fad24 in NIH-3T3 cells promoted adipogenesis in the presence of a ligand for peroxisome proliferator-activated receptor gamma. FAD24 localizes in the nucleus, especially within nuclear speckles. As the nuclear speckle functions as a nascent transcription and pre-mRNA splicing machinery, there is a possibility that FAD24 functions as one of the components for transcription and/or pre-mRNA splicing and positively regulates adipocyte differentiation.

Separate membrane targeting and anchoring domains function in the localization of the S. cerevisiae Cdc24p guanine nucleotide exchange factor.

Toenjes KA, Simpson D, Johnson DI
Curr Genet. 2004; 45: 257-64

The Saccharomyces cerevisiae Cdc24p guanine nucleotide exchange factor (GEF) activates the Cdc42p GTPase to a GTP-bound state. Cdc42p and Cdc24p co-localize at polarized growth sites during the cell cycle; and analysis of Cdc24p carboxyl-terminal truncation and site-specific mutations identified a 56-amino-acid domain as being necessary and sufficient for localization to these sites. This domain, however, was unable to anchor Cdc24p at these sites. Anchoring was restored by fusing the targeting domain to either the Cdc24p carboxyl-terminal PC domain that interacts with the Bem1p scaffold protein or the Cdc42p KKSKKCTIL membrane-anchoring domain. Mutant analysis and protein solubilization data indicated that anchoring required Bem1p, the Rsr1p/Bud1p GTPase, and the potential transmembrane protein YGR221Cp/Tos2p. These data are consistent with Cdc24p localization being a function of both membrane-specific targeting and subsequent anchoring within a multi-protein complex. Given the highly conserved roles of GEFs in Cdc42p signaling pathways, it is likely that similar targeting and anchoring mechanisms exist for Rho GEFs in other eukaryotes.

An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin.

Tange Y, Hirata A, Niwa O
J Cell Sci. 2002; 115: 4375-85

We identified a novel fission yeast gene, ned1(+), with pleiotropic mutations that have a high incidence of chromosome missegregation, aberrantly shaped nuclei, overdeveloped endoplasmic reticulum-like membranes, and increased sensitivity to a microtubule destabilizing agent. Ned1 protein, which was phosphorylated in a growth-related manner, interacted in a yeast two-hybrid system with Dis3 as well as with Pim1/RCC1 (nucleotide exchange factor for Ran). Ned1 also interacted with an essential nucleoporin, a probable homologue of mammalian Nup98/96. The ned1 gene displayed a variety of genetic interactions with factors involved in nuclear transport and chromosome segregation, including the crm1 (exportin), spi1 (small GTPase Ran), pim1, and dis genes. A substitution mutation that affected the two-hybrid interaction with Dis3 increased chromosome instability, suggesting the functional importance of the interaction. Overproduction of Ned1 protein induced formation of an abnormal microtubule bundle within the nucleus, apparently independently of the spindle pole body, but dependent on pim1(+) activity. The ned1(+) gene belongs to an evolutionarily conserved gene family, which includes the mouse Lpin genes, one of whose mutations is responsible for lipodystrophy.

Structure and assembly of the Nup84p complex.

Siniossoglou S, Lutzmann M, Santos-Rosa H, Leonard K, Mueller S, Aebi U, Hurt E
J Cell Biol. 2000; 149: 41-54

The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that a pool of green fluorescent protein-tagged Sec13p localizes to the nuclear pores in vivo, and identify sec13 mutant alleles that are synthetically lethal with nup85Delta and affect the localization of a green fluorescent protein-Nup49p reporter protein. In the electron microscope, sec13 mutants exhibit structural defects in nuclear pore complex (NPC) and nuclear envelope organization. For the assembly of the complex, Nup85p, Nup120p, and Nup145p-C are essential. A highly purified Nup84p complex was isolated from yeast under native conditions and its molecular mass was determined to be 375 kD by quantitative scanning transmission electron microscopy and analytical ultracentrifugation, consistent with a monomeric complex. Furthermore, the Nup84p complex exhibits a Y-shaped, triskelion-like morphology 25 nm in diameter in the transmission electron microscope. Thus, the Nup84p complex constitutes a paradigm of an NPC structural module with distinct composition, structure, and a role in nuclear mRNA export and NPC bio- genesis.

Phospholipid metabolism and nuclear function: roles of the lipin family of phosphatidic acid phosphatases.

Siniossoglou S
Biochim Biophys Acta. 2013; 1831: 575-81

Phospholipids play important roles in nuclear function as dynamic building blocks for the biogenesis of the nuclear membrane, as well as signals by which the nucleus communicates with other organelles, and regulate a variety of nuclear events. The mechanisms underlying the nuclear roles of phospholipids remain poorly understood. Lipins represent a family of phosphatidic acid (PA) phosphatases that are conserved from yeasts to humans and perform essential functions in lipid metabolism. Several studies have identified key roles for lipins and their regulators in nuclear envelope organization, gene expression and the maintenance of lipid homeostasis in yeast and metazoans. This review discusses recent advances in understanding the roles of lipins in nuclear structure and function. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

NBP1 (Nap1 binding protein 1), an essential gene for G2/M transition of Saccharomyces cerevisiae, encodes a protein of distinct sub-nuclear localization.

Shimizu Y, Akashi T, Okuda A, Kikuchi A, Fukui K
Gene. 2000; 246: 395-404

Nap1p is identified in mammalian cell extract by its intrinsic activity to facilitate nucleosome assembly in vitro in the physiological ionic condition. The homologous proteins are present in most eukaryotes, and their functional analyses in vitro have suggested that they are necessary to keep proper nucleosome structures in transcription and replication. This protein is also identified for its interaction with Clb2p in vitro. To address the function of Nap1p in vivo, we have surveyed for proteins to interact with Nap1p by two-hybrid system and obtained two genes, NBP1 and NBP2 (Nap1 Binding Protein 1 and 2). NBP1 is an essential gene and encodes a novel protein consisting of 319 amino acids, with a coiled-coil structure in the center of the predicted amino acid sequence. Several A-kinase dependent phosphorylation sites and Cdc28p kinase-dependent sites are also observed. By isolating the temperature-sensitive mutant, we demonstrate that the nuclear division at a non-permissive temperature is delayed and that the population of cells with a large bud carrying a single nucleus with a short spindle are increased. This mutant also confers resistance against benomyl, a microtubule-destabilizing agent. Judging from the green fluorescent protein (GFP) signal fused with Nbp1p, this protein localizes in the nucleus as one or two tiny dots.

Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins.

Schneiter R, Cole CN
Nucleus. 2010; 1: 387-92

The nuclear envelope harbors numerous large proteinaceous channels, the nuclear pore complexes (NPCs), through which macromolecular exchange between the cytosol and the nucleoplasm occurs. This double-membrane nuclear envelope is continuous with the endoplasmic reticulum and thus functionally connected to such diverse processes as vesicular transport, protein maturation and lipid synthesis. Recent results obtained from studies in Saccharomyces cerevisiae indicate that assembly of the nuclear pore complex is functionally dependent upon maintenance of lipid homeostasis of the ER membrane. Previous work from one of our laboratories has revealed that an integral membrane protein Apq12 is important for the assembly of functional nuclear pores. Cells lacking APQ12 are viable but cannot grow at low temperatures, have aberrant NPCs and a defect in mRNA export. Remarkably, these defects in NPC assembly can be overcome by supplementing cells with a membrane fluidizing agent, benzyl alcohol, suggesting that Apq12 impacts the flexibility of the nuclear membrane, possibly by adjusting its lipid composition when cells are shifted to a reduced temperature. Our new study now expands these findings and reveals that an essential membrane protein, Brr6, shares at least partially overlapping functions with Apq12 and is also required for assembly of functional NPCs. A third nuclear envelope membrane protein, Brl1, is related to Brr6, and is also required for NPC assembly. Because maintenance of membrane homeostasis is essential for cellular survival, the fact that these three proteins are conserved in fungi that undergo closed mitoses, but are not found in metazoans or plants, may indicate that their functions are performed by proteins unrelated at the primary sequence level to Brr6, Brl1 and Apq12 in cells that disassemble their nuclear envelopes during mitosis.

Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.

Schmitt C, von Kobbe C, Bachi A, Pante N, Rodrigues JP, Boscheron C, Rigaut G, Wilm M, Seraphin B, Carmo-Fonseca M, Izaurralde E
EMBO J. 1999; 18: 4332-47

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.

Bringing KASH under the SUN: the many faces of nucleo-cytoskeletal connections.

Razafsky D, Hodzic D
J Cell Biol. 2009; 186: 461-72

The nucleus is the most prominent cellular organelle, and its sharp boundaries suggest the compartmentalization of the nucleoplasm from the cytoplasm. However, the recent identification of evolutionarily conserved linkers of the nucleoskeleton to the cytoskeleton (LINC) complexes, a family of macromolecular assemblies that span the double membrane of the nuclear envelope, reveals tight physical connections between the two compartments. Here, we review the structure and evolutionary conservation of SUN and KASH domain-containing proteins, whose interaction within the perinuclear space forms the "nuts and bolts" of LINC complexes. Moreover, we discuss the function of these complexes in nuclear, centrosomal, and chromosome dynamics, and their connection to human disease.

Regulated nuclear accumulation of the yeast hsp70 Ssa4p in ethanol-stressed cells is mediated by the N-terminal domain, requires the nuclear carrier Nmd5p and protein kinase C.

Quan X, Rassadi R, Rabie B, Matusiewicz N, Stochaj U
FASEB J. 2004; 18: 899-901

Cytoplasmic proteins of the hsp70/hsc70 family redistribute in cells that have been exposed to stress. As such, the hsp70 Ssa4p of the budding yeast S. cerevisiae accumulates in nuclei when cells are treated with ethanol, whereas classical nuclear import is inhibited under these conditions. The N-terminal domain of Ssa4p, which is lacking a classical NLS, mediates nuclear accumulation upon ethanol exposure. Concentration of the Ssa4p N-terminal segment in nuclei is reversible, as the protein relocates to the cytoplasm when cells recover. Mutant analysis demonstrates that the small GTPase Gsp1p and GTPase-modulating factors are required to accumulate Ssa4p in nuclei upon ethanol stress. Moreover, we have identified the importin-beta family member Nmd5p as the nuclear carrier for Ssa4p. Ethanol treatment significantly increases the formation of import complexes containing Nmd5p and the N-terminal Ssa4p domain. Likewise, docking of the carrier Nmd5p at the nuclear pore is enhanced by ethanol. Furthermore, we show that the stressed-induced nuclear accumulation of Ssa4p depends on signaling through protein kinase C and requires sensors of the cell integrity pathway.

Yeast Pex14p possesses two functionally distinct Pex5p and one Pex7p binding sites.

Niederhoff K, Meindl-Beinker NM, Kerssen D, Perband U, Schafer A, Schliebs W, Kunau WH
J Biol Chem. 2005; 280: 35571-8

Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14delta cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane.

Nuclear export receptor Xpo1/Crm1 is physically and functionally linked to the spindle pole body in budding yeast.

Neuber A, Franke J, Wittstruck A, Schlenstedt G, Sommer T, Stade K
Mol Cell Biol. 2008; 28: 5348-58

The spindle pole body (SPB) represents the microtubule organizing center in the budding yeast Saccharomyces cerevisiae. It is a highly structured organelle embedded in the nuclear membrane, which is required to anchor microtubules on both sides of the nuclear envelope. The protein Spc72, a component of the SPB, is located at the cytoplasmic face of this organelle and serves as a receptor for the gamma-tubulin complex. In this paper we show that it is also a binding partner of the nuclear export receptor Xpo1/Crm1. Xpo1 binds its cargoes in a Ran-dependent fashion via a short leucine-rich nuclear export signal (NES). We show that binding of Spc72 to Xpo1 depends on Ran-GTP and a functional NES in Spc72. Mutations in this NES have severe consequences for mitotic spindle morphology in vivo. This is also the case for xpo1 mutants, which show a reduction in cytoplasmic microtubules. In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.

Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region.

Nagai Y, Nogami S, Kumagai-Sano F, Ohya Y
Mol Cell Biol. 2003; 23: 1726-36

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region.

Genome-wide synthetic lethal screens identify an interaction between the nuclear envelope protein, Apq12p, and the kinetochore in Saccharomyces cerevisiae.

Montpetit B, Thorne K, Barrett I, Andrews K, Jadusingh R, Hieter P, Measday V
Genetics. 2005; 171: 489-501

The maintenance of genome stability is a fundamental requirement for normal cell cycle progression. The budding yeast Saccharomyces cerevisiae is an excellent model to study chromosome maintenance due to its well-defined centromere and kinetochore, the region of the chromosome and associated protein complex, respectively, that link chromosomes to microtubules. To identify genes that are linked to chromosome stability, we performed genome-wide synthetic lethal screens using a series of novel temperature-sensitive mutations in genes encoding a central and outer kinetochore protein. By performing the screens using different mutant alleles of each gene, we aimed to identify genetic interactions that revealed diverse pathways affecting chromosome stability. Our study, which is the first example of genome-wide synthetic lethal screening with multiple alleles of a single gene, demonstrates that functionally distinct mutants uncover different cellular processes required for chromosome maintenance. Two of our screens identified APQ12, which encodes a nuclear envelope protein that is required for proper nucleocytoplasmic transport of mRNA. We find that apq12 mutants are delayed in anaphase, rereplicate their DNA, and rebud prior to completion of cytokinesis, suggesting a defect in controlling mitotic progression. Our analysis reveals a novel relationship between nucleocytoplasmic transport and chromosome stability.

The luminal N-terminus of yeast Nvj1 is an inner nuclear membrane anchor.

Millen JI, Pierson J, Kvam E, Olsen LJ, Goldfarb DS
Traffic. 2008; 9: 1653-64

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae is largely divided between perinuclear and cortical compartments. Yeast Nvj1 localizes exclusively to small patches on the perinuclear ER where it interacts with Vac8 in the vacuole membrane to form nucleus-vacuole (NV) junctions. Three regions of Nvj1 mediate the biogenesis of NV junctions. A membrane-spanning domain targets the protein to the ER. The C-terminus binds Vac8 in the vacuole membrane, which induces the clustering of both proteins into NV junctions. The luminal N-terminus is required for strict perinuclear localization. Three-dimensional cryo-electron tomography reveals that Nvj1 clamps the separation between the two nuclear membranes to half the width of bulk nuclear envelope. The N-terminus contains a hydrophobic sequence bracketed by basic residues that resembles outer mitochondrial membrane signal-anchors. The hydrophobic sequence can be scrambled or reversed without affecting function. Mutations that reduce the hydrophobicity of the core sequence or affect the distribution of basic residues cause mislocalization to the cortical ER. We conclude that the N-terminus of Nvj1 is a retention sequence that bridges the perinuclear lumen and inserts into the inner nuclear membrane.

Distinct roles for key karyogamy proteins during yeast nuclear fusion.

Melloy P, Shen S, White E, Rose MD
Mol Biol Cell. 2009; 20: 3773-82

During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.

Domain analysis of the Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein, Nab2p. Dissecting the requirements for Nab2p-facilitated poly(A) RNA export.

Marfatia KA, Crafton EB, Green DM, Corbett AH
J Biol Chem. 2003; 278: 6731-40

Mature poly(A) RNA transcripts are exported from the nucleus in complex with heterogeneous nuclear ribonucleoproteins (hnRNPs). Nab2p is an essential Saccharomyces cerevisiae hnRNP protein that interacts with poly(A) RNA and shuttles between the nucleus and cytoplasm. Functional Nab2p is required for export of poly(A) RNA from the nucleus. The Nab2 protein consists of the following four domains: a unique N-terminal domain, a glutamine-rich domain, an arginine-glycine (RGG) domain, and a zinc finger domain. We generated Nab2p deletion mutants to analyze the contribution of each domain to the in vivo function of Nab2p. We first tested whether the deletion mutants could replace the essential NAB2 gene. We then examined the impact of these mutations on Nab2p localization, poly(A) RNA localization, and association of Nab2p with poly(A) RNA. Our analyses revealed that the N-terminal domain is required for nuclear export of both poly(A) RNA and Nab2p. We confirm that the RGG domain is important for Nab2p import in vivo. Finally, the zinc finger domain is critical for the interaction between Nab2p and poly(A) RNA in vivo. Our data support a model where Nab2p associates with poly(A) RNA in the nucleus through the zinc finger domain and facilitates the export of the poly(A) RNA through protein interactions mediated by the N-terminal domain.

Mutation or deletion of the Saccharomyces cerevisiae RAT3/NUP133 gene causes temperature-dependent nuclear accumulation of poly(A)+ RNA and constitutive clustering of nuclear pore complexes.

Li O, Heath CV, Amberg DC, Dockendorff TC, Copeland CS, Snyder M, Cole CN
Mol Biol Cell. 1995; 6: 401-417

To identify genes whose products play potential roles in the nucleocytoplasmic export of messenger RNA, we isolated temperature-sensitive strains of Saccharomyces cerevisiae and examined them by fluorescent in situ hybridization. With the use of a digoxigen-tagged oligo-(dT)50 probe, we identified those that showed nuclear accumulation of poly(A)+ RNA when cells were shifted to the nonpermissive temperature. We describe here the properties of yeast strains bearing the rat3-1 mutation (RAT-ribonucleic acid trafficking) and the cloning of the RAT3 gene. When cultured at the permissive temperature of 23 degrees C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA after a shift to 37 degrees C for 4 h. In wild-type cells, nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope. Both indirect immunofluorescence analysis and electron microscopy of rat3-1 cells indicated that NPCs were clustered into one or a few regions of the NE in mutant cells. Similar NPC clustering was seen in mutant cells cultured at temperatures between 15 degrees C and 37 degrees C. The RAT3 gene encodes an 1157-amino acid protein without similarity to other known proteins. It is essential for growth only at 37 degrees C. Cells carrying a disruption of the RAT3 gene were very similar to cells carrying the original rat3-1 mutation; they showed temperature-dependent nuclear accumulation of poly(A)+ RNA and exhibited constitutive clustering of NPCs. Epitope tagging of Rat3p demonstrated that it is located at the nuclear periphery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nup133p.

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope.

Libotte T, Zaim H, Abraham S, Padmakumar VC, Schneider M, Lu W, Munck M, Hutchison C, Wehnert M, Fahrenkrog B, Sauder U, Aebi U, Noegel AA, Karakesisoglou I
Mol Biol Cell. 2005; 16: 3411-24

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.

Snf7p, a component of the ESCRT-III protein complex, is an upstream member of the RIM101 pathway in Candida albicans.

Kullas AL, Li M, Davis DA
Eukaryot Cell. 2004; 3: 1609-18

The success of Candida albicans as an opportunistic pathogen is based in part on its ability to adapt to diverse environments. The RIM101 pathway governs adaptation to neutral-alkaline environments and is required for virulence. Analysis of a genomic two-hybrid study conducted with Saccharomyces cerevisiae revealed that components involved in multivesicular bodies (MVB) transport may interact with RIM101 pathway members. Thus, we hypothesized that these proteins may function in the RIM101 pathway in C. albicans. We identified C. albicans homologs to S. cerevisiae Snf7p, Vps4p, and Bro1p and generated mutants in the cognate gene. We found that snf7Delta/Delta mutants, but not vps4Delta/Delta nor bro1Delta/Delta mutants, had phenotypes similar to, but more severe than, those of RIM101 pathway mutants. We found that the constitutively active RIM101-405 allele partially rescued snf7Delta/Delta mutant phenotypes. The vps4Delta/Delta mutant had subtle phenotypes, but these were not rescued by the RIM101-405 allele. Further, we found that the snf7Delta/Delta, vps4Delta/Delta, and bro1Delta/Delta mutants did not efficiently localize the vital dye FM4-64 to the vacuole and that it was often accumulated in an MVB-like compartment. This phenotype was not rescued by RIM101-405 or observed in RIM101 pathway mutants. These results suggest that Snf7p may serve two functions in the cell: one as a RIM101 pathway member and one for MVB transport to the vacuole.

Dynamic localization of the nuclear import receptor and its interactions with transport factors.

Koepp DM, Wong DH, Corbett AH, Silver PA
J Cell Biol. 1996; 133: 1163-76

Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin-beta, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta. Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal-bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins.

Klupp BG, Granzow H, Fuchs W, Keil GM, Finke S, Mettenleiter TC
Proc Natl Acad Sci U S A. 2007; 104: 7241-6

Although the nuclear envelope is a dynamic structure that disassembles and reforms during mitosis, the formation of membranous vesicles derived from the nuclear envelope has not yet been described in noninfected cells. However, during herpesvirus maturation, intranuclear capsids initiate transit to the cytosol for final maturation by budding at the inner nuclear membrane. Two conserved herpesvirus proteins are required for this primary envelopment, designated in the alphaherpesviruses as pUL31 and pUL34. Here, we show that simultaneous expression of pUL31 and pUL34 of the alphaherpesvirus pseudorabies virus in stably transfected rabbit kidney cells resulted in the formation of vesicles in the perinuclear space that resemble primary envelopes without a nucleocapsid. They contain pUL31 and pUL34 as shown by immunolabeling and are derived from the nuclear envelope. Thus, coexpression of only two conserved herpesvirus proteins without any other viral factor is sufficient to induce the formation of vesicles from the nuclear membrane. This argues for the contribution of cellular factors in this process either recruited from their natural cytoplasmic location or not yet identified as components of the nuclear compartment.

Ribosome biogenesis factors working with a nuclear envelope SUN domain protein: new players in the solar system.

Horigome C, Mizuta K
Nucleus. 2012; 3: 22-8

The nucleolus, the most prominent structure observed in the nucleus, is often called a "ribosome factory." Cells spend an enormous fraction of their resources to achieve the mass-production of ribosomes required by rapid growth. On the other hand, ribosome biogenesis is also tightly controlled, and must be coordinated with other cellular processes. Ribosomal proteins and ribosome biogenesis factors are attractive candidates for this link. Recent results suggest that some of them have functions beyond ribosome biogenesis. Here we review recent progress on ribosome biogenesis factors, Ebp2 and Rrs1, in yeast Saccharomyces cerevisiae. In this organism, Ebp2 and Rrs1 are found in the nucleolus and at the nuclear periphery. At the nuclear envelope, these proteins interact with a membrane-spanning SUN domain protein, Mps3, and play roles in telomere clustering and silencing along with the silent information regulator Sir4. We propose that a protein complex consisting Ebp2, Rrs1 and Mps3 is involved in a wide range of activities at the nuclear envelope.

Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p.

Ho AK, Shen TX, Ryan KJ, Kiseleva E, Levy MA, Allen TD, Wente SR
Mol Cell Biol. 2000; 20: 5736-48

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Delta108 mutant after growth at 37 degrees C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.

Nuclear envelope phosphatase 1-regulatory subunit 1 (formerly TMEM188) is the metazoan Spo7p ortholog and functions in the lipin activation pathway.

Han S, Bahmanyar S, Zhang P, Grishin N, Oegema K, Crooke R, Graham M, Reue K, Dixon JE, Goodman JM
J Biol Chem. 2012; 287: 3123-37

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Deltaspo7Delta strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

Components of coated vesicles and nuclear pore complexes share a common molecular architecture.

Devos D, Dokudovskaya S, Alber F, Williams R, Chait BT, Sali A, Rout MP
PLoS Biol. 2004; 2: 380-380

Numerous features distinguish prokaryotes from eukaryotes, chief among which are the distinctive internal membrane systems of eukaryotic cells. These membrane systems form elaborate compartments and vesicular trafficking pathways, and sequester the chromatin within the nuclear envelope. The nuclear pore complex is the portal that specifically mediates macromolecular trafficking across the nuclear envelope. Although it is generally understood that these internal membrane systems evolved from specialized invaginations of the prokaryotic plasma membrane, it is not clear how the nuclear pore complex could have evolved from organisms with no analogous transport system. Here we use computational and biochemical methods to perform a structural analysis of the seven proteins comprising the yNup84/vNup107-160 subcomplex, a core building block of the nuclear pore complex. Our analysis indicates that all seven proteins contain either a beta-propeller fold, an alpha-solenoid fold, or a distinctive arrangement of both, revealing close similarities between the structures comprising the yNup84/vNup107-160 subcomplex and those comprising the major types of vesicle coating complexes that maintain vesicular trafficking pathways. These similarities suggest a common evolutionary origin for nuclear pore complexes and coated vesicles in an early membrane-curving module that led to the formation of the internal membrane systems in modern eukaryotes.

Nuclear export of the oncoprotein v-ErbA is mediated by acquisition of a viral nuclear export sequence.

DeLong LJ, Bonamy GM, Fink EN, Allison LA
J Biol Chem. 2004; 279: 15356-67

v-ErbA, an oncogenic derivative of the thyroid hormone receptor alpha (TRalpha) carried by the avian erythroblastosis virus, contains several alterations including fusion of a portion of avian erythroblastosis virus Gag to its N terminus, N- and C-terminal deletions, and 13 amino acid substitutions. Nuclear export of v-ErbA occurs through a CRM1-mediated pathway. In contrast, nuclear export of TRalpha and another isoform, TRbeta, is CRM1-independent. To determine which amino acid changes in v-ErbA confer CRM1-dependent nuclear export, we expressed a panel of green and yellow fluorescent protein-tagged mutant and chimeric proteins in mammalian cells. The sensitivity of subcellular trafficking of these mutants to leptomycin B (LMB), a specific inhibitor of CRM1, was assessed by fluorescence microscopy. Our data showed that a nuclear export sequence resides within a 70-amino acid domain in the C-terminal portion of the p10 region of Gag, and in vitro binding assays demonstrated that Gag interacts directly with CRM1. However, a panel of ligand-binding domain mutants of v-ErbA lacking the Gag sequence exhibited greater nuclear localization in the presence of LMB, suggesting that the various amino acid substitutions/deletions may cause a conformation shift, unmasking an additional CRM1-dependent nuclear export sequence. In contrast, the altered DNA-binding domain of the oncoprotein did not contribute to CRM1-dependent nuclear export. Heterokaryon experiments revealed that v-ErbA did not undergo nucleocytoplasmic shuttling when the CRM1 export pathway was blocked by LMB treatment, suggesting that the ability to follow the export pathway used by TRalpha has been lost by the oncoprotein during its evolution. Our findings thus point to the intriguing possibility that acquisition of altered nuclear export capabilities contributes to the oncogenic properties of v-ErbA.

Tissue specificity in the nuclear envelope supports its functional complexity.

de Las Heras JI, Meinke P, Batrakou DG, Srsen V, Zuleger N, Kerr AR, Schirmer EC
Nucleus. 2013; 4: 460-77

Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution.

An essential nuclear envelope integral membrane protein, Brr6p, required for nuclear transport.

de Bruyn Kops A, Guthrie C
EMBO J. 2001; 20: 4183-93

Despite rapid advances in our understanding of the function of the nuclear pore complex in nuclear transport, little is known about the role the nuclear envelope itself may play in this critical process. A small number of integral membrane proteins specific to the envelope have been identified in budding yeast, however, none has been reported to affect transport. We have identified an essential gene, BRR6, whose product, Brr6p, behaves like a nuclear envelope integral membrane protein. Notably, the brr6-1 mutant specifically affects transport of mRNA and a protein reporter containing a nuclear export signal. In addition, Brr6p depletion alters nucleoporin distribution and nuclear envelope morphology, suggesting that the protein is required for the spatial organization of nuclear pores. BRR6 interacts genetically with a subset of nucleoporins, and Brr6-green fluorescent protein (GFP) localizes in a punctate nuclear rim pattern, suggesting location at or near the nuclear pore. However, Brr6-GFP fails to redistribute in a (Delta)nup133 mutant, distinguishing Brr6p from known proteins of the pore membrane domain. We hypothesize that Brr6p is located adjacent to the nuclear pore and interacts functionally with the pore and transport machinery.

Yeast importin-alpha (Srp1) performs distinct roles in the import of nuclear proteins and in targeting proteasomes to the nucleus.

Chen L, Madura K
J Biol Chem. 2014; 289: 32339-52

Srp1 (importin-alpha) can translocate proteins that contain a nuclear localization signal (NLS) into the nucleus. The loss of Srp1 is lethal, although several temperature-sensitive mutants have been described. Among these mutants, srp1-31 displays the characteristic nuclear import defect of importin-alpha mutants, whereas srp1-49 shows a defect in protein degradation. We characterized these and additional srp1 mutants to determine whether distinct mechanisms were required for intracellular proteolysis and the import of NLS-containing proteins. We determined that srp1 mutants that failed to import NLS-containing proteins (srp1-31 and srp1-55) successfully localized proteasomes to the nucleus. In contrast, srp1 mutants that did not target proteasomes to the nucleus (srp1-49 and srp1-E402Q) were able to import NLS-containing proteins. The proteasome targeting defect of specific srp1 mutants caused stabilization of nuclear substrates and overall accumulation of multiubiquitylated proteins. Co-expression of a member of each class of srp1 mutants corrected both the proteasome localization defect and the import of NLS-containing proteins. These findings indicate that the targeting of proteasomes to the nucleus occurs by a mechanism distinct from the Srp1-mediated import of nuclear proteins.

The SUN protein Mps3 controls Ndc1 distribution and function on the nuclear membrane.

Chen J, Smoyer CJ, Slaughter BD, Unruh JR, Jaspersen SL
J Cell Biol. 2014; 204: 523-39

In closed mitotic systems such as Saccharomyces cerevisiae, nuclear pore complexes (NPCs) and the spindle pole body (SPB) must assemble into an intact nuclear envelope (NE). Ndc1 is a highly conserved integral membrane protein involved in insertion of both complexes. In this study, we show that Ndc1 interacts with the SUN domain-containing protein Mps3 on the NE in live yeast cells using fluorescence cross-correlation spectroscopy. Genetic and molecular analysis of a series of new ndc1 alleles allowed us to understand the role of Ndc1-Mps3 binding at the NE. We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication. Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component. Based on our data we propose that the Ndc1-Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB.

Efficient nuclear transport of structurally disturbed cargo: mutations in a cargo protein switch its cognate karyopherin.

Chaves SR, Rosenblum JS
PLoS One. 2011; 6: 16846-16846

The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus.

Establishment of the ambient pH signaling complex in Aspergillus nidulans: PalI assists plasma membrane localization of PalH.

Calcagno-Pizarelli AM, Negrete-Urtasun S, Denison SH, Rudnicka JD, Bussink HJ, Munera-Huertas T, Stanton L, Hervas-Aguilar A, Espeso EA, Tilburn J, Arst HN Jr, Penalva MA
Eukaryot Cell. 2007; 6: 2365-75

The Aspergillus nidulans ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a three TMD (3-TMD) protein with an N-terminal signal peptide, and we show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27-kDa form, but its absence markedly reduces the accumulation of the 53-kDa intermediate after cells are shifted to an alkaline pH. PalI and its homologues contain a predicted luminal, conserved Gly-Cys-containing motif that distantly resembles a Gly-rich dimerization domain. The Gly44Arg and Gly47Asp substitutions within this motif lead to loss of function. The Gly47Asp substitution prevents plasma membrane localization of PalI-green fluorescent protein (GFP) and leads to its missorting into the multivesicular body pathway. Overexpression of the likely ambient alkaline pH receptor, the 7-TMD protein PalH, partially suppresses the null palI32 mutation. Although some PalH-GFP localizes to the plasma membrane, it predominates in internal membranes. However, the coexpression of PalI to stoichiometrically similar levels results in the strong predominance of PalH-GFP in the plasma membrane. Thus, one role for PalI, but possibly not the only role, is to assist with plasma membrane localization of PalH. These data, considered along with previous reports for both Saccharomyces cerevisiae and A. nidulans, strongly support the prevailing model of pH signaling involving two spatially segregated complexes: a plasma membrane complex containing PalH, PalI, and the arrestin-like protein PalF and an endosomal membrane complex containing PalA and PalB, to which PacC is recruited for its proteolytic activation.

Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins.

Brownawell AM, Macara IG
J Cell Biol. 2002; 156: 53-64

We have identified a novel human karyopherin (Kap) beta family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.

Crm1-mediated nuclear export of the Schizosaccharomyces pombe transcription factor Cuf1 during a shift from low to high copper concentrations.

Beaudoin J, Labbe S
Eukaryot Cell. 2007; 6: 764-75

In this study, we examine the fate of the nuclear pool of the Schizosaccharomyces pombe transcription factor Cuf1 in response to variations in copper levels. A nuclear pool of Cuf1-green fluorescent protein (GFP) was generated by expressing a functional cuf1(+)-GFP allele in the presence of a copper chelator. We then extinguished cuf1(+)-GFP expression and tracked the changes in the localization of the nuclear pool of Cuf1-GFP in the presence of low or high copper concentrations. Treating cells with copper as well as silver ions resulted in the nuclear export of Cuf1. We identified a leucine-rich nuclear export signal (NES), (349)LAALNHISAL(358), within the C-terminal region of Cuf1. Mutations in this sequence abrogated Cuf1 export from the nucleus. Furthermore, amino acid substitutions that impair Cuf1 NES function resulted in increased target gene expression and a concomitant cellular hypersensitivity to copper. Export of the wild-type Cuf1 protein was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export protein Crm1. We further show that cells expressing a temperature-sensitive mutation in crm1(+) exhibit increased nuclear accumulation of Cuf1 at the nonpermissive temperature. Although wild-type Cuf1 is localized in the nucleus in both conditions, we observed that the protein can still be inactivated by copper, resulting in the repression of ctr4(+) gene expression in the presence of exogenous copper. These results demonstrate that nuclear accumulation of Cuf1 per se is not sufficient to cause the unregulated expression of the copper transport genes like ctr4(+). In addition to nuclear localization, a functional Cys-rich domain or NES element in Cuf1 is required to appropriately regulate copper transport gene expression in response to changes in intracellular copper concentration.

Protein quality control: Nuclear membrane proteins in check.

Baumann K
Nat Rev Mol Cell Biol. 2014; 15: 700-700

Functional analysis of Tpr: identification of nuclear pore complex association and nuclear localization domains and a role in mRNA export.

Bangs P, Burke B, Powers C, Craig R, Purohit A, Doxsey S
J Cell Biol. 1998; 143: 1801-12

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe.

Azad AK, Tani T, Shiki N, Tsuneyoshi S, Urushiyama S, Ohshima Y
Mol Biol Cell. 1997; 8: 825-41

Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.

The Saccharomyces cerevisiae RanGTP-binding protein msn5p is involved in different signal transduction pathways.

Alepuz PM, Matheos D, Cunningham KW, Estruch F
Genetics. 1999; 153: 1219-31

In eukaryotes, control of transcription by extracellular signals involves the translocation to the nucleus of at least one component of the signal transduction pathway. Transport through the nuclear envelope requires the activity of an import or export receptor that interacts with the small GTPase Ran. We have cloned the MSN5 gene of the yeast Saccharomyces cerevisiae that is postulated to encode one of these receptors. Msn5p belongs to a family of proteins with a conserved N-terminal sequence that acts as a RanGTP-binding domain. The results presented here provide genetic data supporting Msn5p involvement in several different signal transduction pathways. All of these pathways include changes in gene expression, and regulated nucleocytoplasmic redistribution of a component in response to external conditions has already been described in some of them. We have cloned MSN5 following two different strategies. Msn5p was constitutively localized in the nucleus. Phenotypic analysis of the msn5 mutant demonstrated that this protein participates in processes such as catabolite repression, calcium signaling, mating, and cell proliferation, as well as being involved in previously characterized phosphate utilization. Therefore, Msn5p could be a receptor for several proteins involved in different signaling pathways.

Kap104p: a karyopherin involved in the nuclear transport of messenger RNA binding proteins.

Aitchison JD, Blobel G, Rout MP
Science. 1996; 274: 624-7

A cytosolic yeast karyopherin, Kap104p, was isolated and shown to function in the nuclear import of a specific class of proteins. The protein bound directly to repeat-containing nucleoporins and to a cytosolic pool of two nuclear messenger RNA (mRNA) binding proteins, Nab2p and Nab4p. Depletion of Kap104p resulted in a rapid shift of Nab2p from the nucleus to the cytoplasm without affecting the localization of other nuclear proteins tested. This finding suggests that the major function of Kap104p lies in returning mRNA binding proteins to the nucleus after mRNA export.